The goal of this project is the development of therapeutic radiopharmaceuticals based on targeting the decay of Auger-electron-emitting radioisotopes to specific sequences in DNA (genes) using triplex-forming oligonucleotides (TFOs) as delivery vehicles. In in vitro studies we have demonstrated that TFOs are able to deliver Auger electron emitters to specific targets in cellular DNA in order to inactivate genes and/or kill the cells containing the target sequences. Decay of I-125 in TFOs results in strand breaks in both strands of the target DNA with an efficiency from 0.4-0.8 break/decay. Higher efficiency can be achieved with radionuclide multiple labeling. Breaks are confined to the triplex target sequence, and 90 percent of the sequence-specific breaks are located within 10 bp around the decay site. We showed that radiotoxicity of TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated I-125UdR. TFOs were designed to target the human MDR1 gene that is amplified in KB-VI cells in culture. The TFOs were labeled with I-125, and the targeting was detected by the presence of radioiodine-induced breaks. The breaks were found in DNA purified from I-125-TFO-treated isolated nuclei and digitonin-permeabelized cells. To increase the efficiency of targeting, a new generation of chemically modified oligonucleotides with increased in vivo stability permitting one-step labeling with Auger electron emitters is being developed. We have developed a rapid procedure for incorporation of the short life Auger electron emitters I-123 and I-111In-111 into ODNs and demonstrated that decay of these more clinically relevant radioisotopes produces DNA breaks with a yield comparable to that of I-125. We also have shown that the fine structure of DNA damage by decay of Auger electron emitter depends on local DNA conformation and that by analyzing the DNA damage, one can obtain information on the structure of DNA in nucleoprotein complexes both in vitro and in vivo. Based on this principle, a new method of radioprobing DNA-protein complexes has been demonstrated in several model systems. In addition, studies have been initiated to investigate the mechanisms of Auger-electron?induced DNA strand break repair in human cells. We have developed efficient methods of producing and isolating specific forms (form I and form II) of damaged shuttle vector plasmid DNA, using both oxidative agents and TFO-bound Auger-emitting radionuclides as damaging agents. A liposome delivery system has been developed for efficient delivery of damaged DNA into human cells in order to evaluate the in vivo repairability and mutagenicity of site-specific DNA double strand breaks (DSBs) induced by I-125-labeled TFOs. Using the methods described above, I-125-TFO-induced DNA DSBs were found to be very effective at inactivating a shuttle-vector-borne target reporter gene by mutagenic disruption. The mutation frequency for I-125-TFO-induced DSB was approximately 80 percent, and the mutation spectrum was dominated by multiple base deletions involving the targeted I-125 decay site. The I-125-TFO-induced DSB was also approximately 100 times more refractory to repair than oxidatively induced DSB similar to those produced by ionizing radiation and reactive oxygen species (ROS) such as hydroxyl radicals. In vitro DSB repair assays have been developed to permit isolation of human proteins that are involved in DSB repair and to analyze DNA reaction products at the molecular level for comparison to DNA repaired in vivo. This assay employs plasmid DNA containing a DSB similar to that produced by ionizing radiation and other ROS. This DSB lesion more closely models naturally occurring DSB than DSB produced by other methods, such as restriction enzymes. In support of this assay, methods have been developed to produce and recover the large quantities of plasmid substrate DNA (linearized by bleomycin) necessary for chromatography and biochemistry procedures. The assay has been optimized for DSB rejoining using human HeLa cell extracts. Optimal conditions depend on the complexity of DSB introduced into the substrate DNA, with slight variations of pH and ionic strength being the variables. Standard reaction conditions have been established, and, under these conditions, the initial-repair-reaction rate for complex DSB produced by bleomycin is approximately twofold less than for the equivalent, but less chemically complex, restriction-enzyme-produced DSB. The goals of the studies outlined above are to identify the human repair pathways involved in Auger-emitter-induced DSB repair; assess the consequences of repairing these lesions; and examine methods by which these repair processes can be manipulated to augment the radiotherapeutic effects of TFOs labeled with Auger-electron-emitting radionuclides.